Proc. Nail. Acad. Sci. USA Vol. 89, pp. 10721-10725, November 1992 Pharmacology

Non--derived (F2-isoprostanes) are formed in situ on phospholipids (/lipids/oxidative stress/peroxidation/free radicals) JASON D. MORROW, JOSEPH A. AWAD, HOLLIS J. BOSS, IAN A. BLAIR, AND L. JACKSON ROBERTS II* Departments of Pharmacology and Medicine, Vanderbilt University, Nashville, TN 37232.6602 Communicated by Philip Needleman, July 21, 1992 (receivedfor review March 11, 1992)

ABSTRACT We recently reported the discovery ofa series the formation ofthese prostanoids occurs independent ofthe of bioactive F2-like compounds (F2-isoprostanes) catalytic activity of the cyclooxygenase enzyme, which had that are produced in vivo by free radical-catalyzed peroxidation been considered obligatory for endogenous bio- ofarachidonic acid independent ofthe cyclooxygenase enzyme. synthesis. Circulating levels ofthese compounds were shown Inasmuch as phospholipids readily undergo peroxidation, we to increase dramatically in animal models of free radical examined the possibility that F2-isoprostanes may be formed in injury (8). Interestingly, the levels of these prostanoids in situ on phospholipids. Initial support for this hypothesis was normal human plasma and urine are one or two orders of obtained by the rmding that levels of free F2-isoprostanes magnitude higher than those of produced by measured after hydrolysis oflipids extracted from livers ofrats the cyclooxygenase enzyme. Formation ofthese compounds treated with CCI4 to induce lipid peroxidation were more than proceeds through intermediates composed of four positional 100-fold higher than levels in untreated animal. Further, peroxyl radical isomers of which undergo increased levels of lipid-associated F2-isoprostanes in livers of endocyclization to yield bicyclic endoperoxide PGG2-like CCI4-treated rats preceded the appearance of free compounds compounds. The PGG2-like compounds are then reduced to in the circulation, suggesting that the free compounds arose PGF2-like compounds (9). In conjunction with the Committee from hydrolysis of peroxidized lipids. This concept was sup- of Nomenclature constituted by the Joint Com- ported by demonstrating that free F2-isoprostanes were re- mission on Biochemical Nomenclature, a facile nomencla- leased after incubation of lipid extracts with bee venom phos- ture for the individual compounds produced by this mecha- pholipase A2 in vitro. When these lipid extracts were analyzed nism is being developed, based on the general term "iso- by HPLC, fractions that yielded large quantities of free F2- prostanes" and the type of prostane ring they contain. isoprostanes after hydrolysis eluted at a much more polar Unsaturated fatty acids in phospholipids readily undergo retention volume than nonoxidized phosphatidylcholine. Anal- peroxidation upon exposure to free radicals. Peroxidation of ysis of these polar lipids by fast atom bombardment mass arachidonoyl phospholipids would result in the formation of spectrometry established that they were F2-isoprostane- the positional peroxyl isomers of arachidonic acid that are containing species of phosphatidylcholine. Thus, unlike cy- intermediates leading to the formation of isoprostanes (9). clooxygenase-derived prostanoids, F2-isoprostanes are initially Therefore, we investigated the possibility that isoprostanes formed in situ on phospholipids, from which they are subse- may be formed completely in situ on phospholipids. quently released preformed, presumably by phospholipases. Molecular modeling of FI-isoprostane-containing phospholip- ids reveals them to be remarkably distorted molecules. Thus, EXPERIMENTAL PROCEDURES the formation of these phospholipid species in lipid bilayers Analysis of F2-Isoprostanes. Purification, derivatization, may contribute in an important way to alterations in fluidity and analysis ofF2-isoprostanes by gas chromatography/mass and integrity of cellular membranes, well-known sequelae of spectrometry were performed as described (8, 9). oxidant is ury. Animal Model of Free Radical-Induced Lipid Peroxidation. Endogenous lipid peroxidation was induced in rats by ad- Free radicals derived from oxygen are thought to play an ministration ofCC4 as described (10). At various time points, important role in the pathophysiology ofan increasingly wide animals were sacrificed and the livers were removed, frozen variety ofhuman diseases (1-4). Nonetheless, much remains in liquid N2, and either processed immediately or stored at to be understood about mechanisms ofoxidant injury in vivo. -70°C. We have previously determined that formation of Polyunsaturated fatty acid-containing lipids are recognized F2-isoprostanes by autoxidation of lipids in vitro is com- targets of oxidant damage, readily undergoing peroxidation pletely inhibited at -70°C. upon exposure to free radicals. Peroxidation of lipids can Extraction, Purification, and Hydrolysis of Lipids. Lipids greatly alter the physicochemical properties of membrane from livers were extracted (11), subjected to alkaline hydro- lipid bilayers, resulting in severe cellular dysfunction. In lysis, and subsequently analyzed for F2-isoprostanes as de- addition, a variety of lipid byproducts are produced as a scribed (9). Depending on the experiment, 0.005% butylated consequence of lipid peroxidation, some of which can exert hydroxytoluene or 0.5% triphenylphosphine was added to adverse biological effects (5-7). lipid extracts during the extraction procedure. Recently, we reported the discovery that a series of Release of F2rsoprostanes from Lipid Extracts by Apis prostaglandin (PG)-like compounds capable of exerting po- melifera Venom Phospholipase A2. Lipid extracts of livers of tent biological activity are produced in vivo in humans as CCL4-treated rats (containing approximately 1 ,mol of phos- products of free radical-catalyzed peroxidation of arachi- pholipid) were allowed to react with Apis mellifera venom donic acid (8). The notable aspect of this discovery was that phospholipase A2 (approximately 200 ,g) (Sigma) as de- scribed (12). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: PG, prostaglandin. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 10721 Downloaded by guest on September 29, 2021 10722 Pharmacology: Morrow et al. Proc. Natl. Acad Sci. USA 89 (1992) As a positive control for enzymatic activity, phosphatidyl- tanes measured after the hydrolysis of lipid extracts from choline containing [3H]arachidonate in the sn-2 position livers of rats 2 hr after administration of CCL1 (2 ml/kg) were (New England Nuclear) was added to the incubation mixture 825 + 198 ng/g of liver (n = 5). These levels were 142-fold and the percent of radiolabeled arachidonate released was higher than levels measured after saponification of lipid determined. After 2 hr, the diethyl ether was evaporated and extracts of livers of untreated rats (5.8 + 1.2 ng/g of liver) (n free arachidonic acid and phospholipids were extracted from = 5). Levels of free F2-isoprostanes measured in lipid ex- 1/2 of the incubation mixture at pH 7 with CHC13/methanol tracts that were not subjected to saponification were <2% of (2:1, vol/vol) (13). The organic layer was then evaporated levels measured after saponification. under a stream of N2 and the residue was subjected to TLC The following evidence was obtained confirming that the (silica gel 60A LK6D, Whatman) using a solvent system of compounds detected after alkaline hydrolysis of the lipid petroleum ether/diethyl ether/acetic acid (90:10:1, vol/vol). extracts of liver were identical to F2-isoprostanes previously Free arachidonic acid migrates with an Rf of 0.18 and identified in human plasma and urine: (i) analysis of the phospholipids remain at the origin. The ratio of free arachi- compounds by gas chromatography/mass spectrometry as a donic acid to esterified phospholipid arachidonate was de- deuteriated silyl ether derivative indicated the presence of termined by comparing the amount of radioactivity present three hydroxyl groups; (ii) analysis after catalytic hydroge- between Rf 0.15-0.25 to that present at the origin. nation indicated the presence of two double bonds; (iii) all Free F2-isoprostanes were extracted from the remaining 1/2 compounds formed cyclic boronate derivatives, indicating of the reaction mixture with an equal volume of ethyl acetate the presence of cis prostane ring hydroxyl groups; and finally twice after decreasing the pH to 3 with 1 M HCl. After (iv) analysis by electron ionization mass spectrometry re- evaporation of the organic layer under N2, the residue was vealed multiple compounds eluting from the capillary GC resuspended in 0.5 ml of methanol to which 10 ml of pH 3 column over approximately a 40-sec period that yielded mass water was added. Free F2-isoprostanes were then reextracted spectra essentially identical to those obtained previously for by using C18 Sep-paks (Waters), purified by TLC, and F2-isoprostanes (data not shown) (8, 9). quantified by mass spectrometry as described (8). The per- The above data were consistent with the hypothesis that cent of total F2-isoprostanes released by bee venom phos- F2-isoprostanes are present in vivo in ester linkages to lipids. pholipase A2 was calculated by determining the ratio of the However, we also considered the possibility that their for- amount of free F2-isoprostanes released by enzymatic hy- mation may not have occurred actually in vivo but rather ex drolysis to the amount released by chemical hydrolysis of an vivo during or after lipid extraction and saponification from aliquot (approximately 1 ,umol) of the same lipid extract. peroxyl radical precursors of arachidonic acid present on Microsomal CoA Incubation Experiments. In these exper- oxidized lipids. To test this possibility, lipids were treated iments, esterification of tritiated PGF2,, into lysophosphati- during extraction prior to hydrolysis with triphenylphosphine dylcholine by a rat liver microsomal system (14) was com- (5 mg/ml) to reduce peroxyl groups to hydroxyl groups, pared to that of tritiated arachidonic acid. Free and esterified which are incapable of cyclizing to form the prostane ring. If arachidonate were extracted (14) and the ratio was deter- the formation of the F2-isoprostanes that were detected mined as described for the bee venom experiments discussed occurred ex vivo from peroxidized arachidonic acid precur- above. The ratio of free to incorporated PGF2a was deter- sors present in the extracted lipids, then treatment of the mined as for arachidonic acid except that extracts were lipids with triphenylphosphine during extraction to reduce chromatographed in a solvent system of acetone/hexane/ peroxyl groups on arachidonic acid to hydroxyl groups would acetic acid (75:25:1, vol/vol). In this solvent system, phos- markedly decrease the levels of free isoprostanes measured pholipids remain at the origin and PGF2,, has a Rf of 0.30. after saponification. However, the levels of free F2- Analysis of Lipid Extracts from Livers of CC14-Treated Rats isoprostanes measured after hydrolysis of untreated lipid by Normal-Phase HPLC. Normal-phase HPLC analysis of extracts of rat livers obtained 1 hr after administration ofCC14 lipid extracts was performed on a 25 cm x 4.6 mm Econosil (1 ml/kg) (370 ± 121 ng/g ofliver, n = 5) were not suppressed SI column with 5-,um particles (Alltech Associates), using an by pretreatment with triphenylphosphine (353 ± 70 ng/g, n = isocratic solvent system of hexane/isopropyl alcohol/water 5). Further, the presence of the free radical scavenger bu- (4:6:1, vol/vol) at a flow rate of 1 ml/min. UV absorbance tylated hydroxytoluene (BHT; 0.005%) did not reduce levels was monitored continuously at 205 nm. Aliquots of fractions of F2-isoprostanes measured (371 ± 137 ng/g, n = 5). Since eluted were subjected to alkaline hydrolysis, after which free BHT markedly suppresses the formation offree isoprostanes F2-isoprostanes were quantified as described previously. by autoxidation in vitro (9), this result provided additional Analysis of Phospholipids by Fast Atom Bombardment Mass evidence that the formation of the isoprostanes detected did Spectrometry. Fast atom bombardment mass spectra were not occur ex vivo. obtained on a VG 70/250-HF double-focusing mass spec- We then examined the time course of the increase in trometer (VG Instruments, Danvers, MA). A saddle field gun F2-isoprostanes measured in hydrolyzed lipid extracts from was used at 8 kV with xenon as reactant gas at a filament livers of CCI4-treated rats in relation to the appearance of current of 1.2 mA. Negative-ion and positive-ion mass spec- increased levels of free compounds in the circulation. tra were obtained with a matrix of triethanolamine/dimethyl Whereas lipid-associated levels ofF2-isoprostanes in the liver sulfoxide (1:1, vol/vol) at accelerating voltages of 5.5 and 6.0 were found to increase very rapidly, reaching approximately kV, respectively. half maximum as early as 15 min, the appearance ofincreased quantities of free compounds in the circulation was delayed considerably (Fig. 1). This finding was consistent with the RESULTS concept that these compounds are initially formed in situ on Indirect Evidence for Free Radical-Induced Formation of arachidonoyl-containing lipids and subsequently released in F2-Isoprostane-Containing Lipids in Vivo. Initial evidence free form into the circulation. supporting the hypothesis that F2-isoprostanes may be pre- Evidence supporting the above hypothesis was obtained by sent as the acyl groups of lipids in vivo was obtained by demonstrating that incubation of lipid extracts with phos- comparing levels of free F2-isoprostanes in saponified lipid pholipase A2 in vitro results in the release of free F2- extracts of livers from CC4-treated rats and untreated rats. isoprostanes. Incubation of lipid extracts from livers of The liver is the major target organ of CCI toxicity owing to CCI4-treated rats with phospholipase A2 in vitro for 2 hr its high content ofcytochrome P450, which metabolizes CCI4 resulted in a release of free F2-isoprostanes that was 48% ± to trichloromethyl radicals (15). Levels of free F2-isopros- 11% ofthat from chemical saponification (n = 5). As a control Downloaded by guest on September 29, 2021 Pharmacology: Morrow et al. Proc. Nati. Acad. Sci. USA 89 (1992) 10723

Cu r 1000 E C E - 900 > - 800 6 0. - 700 M co 0) .600 CDC_ a, - 500 to 2 a) 400 Cu C CL 300 8 0n aC,0 . 200 g ca 0. 100Nn 0 U-L .. 400 U- 0 2 4 6 8 10 12 14 16 18 20 22 24 6ow0 B Time after CC14 administration, hr MO 20- cm FIG. 1. Time course of appearance of lipid-associated F2- L1 isoprostanes in liver (e) in comparison with the appearance of free 0 10 20 30 40 50 60 compounds in the circulation (m) of rats after intragastric adminis- Elution time, min tration of CC14 (1 ml/kg). Each time point represents the mean ± SD of levels measured in at least four animals. FIG. 2. Normal-phase HPLC chromatogram obtained from the analysis of lipid extracts from livers of CCL1-treated rats. The for enzymatic activity, the extent of hydrolysis of [3H]ara- isocratic solvent system of hexane/isopropyl alcohol/water (4:6:1, chidonate from [3H]arachidonoyl phosphatidylcholine was vol/vol) was used at a flow rate of 1 ml/min. UV absorbance was determined; it was 95% ± 10%o (n = 5). The relevance of the continually monitored at 205 nm. The large UV peak eluted between finding that F2-isoprostane phospholipids were hydrolyzed 18 and 26 ml in chromatogram A represents nonoxidized phosphati- somewhat less efficiently than arachidonic acid phospholip- dylcholine, while the peaks eluted near the solvent front between 2 and 9 ml represent less polar phospholipids and neutral lipids. In ids under the conditions used in vitro may be difficult to chromatogram B is shown the quantities of free F2-isoprostanes interpret in relation to the situation in vivo. This is because measured after alkaline hydrolysis of aliquots of collected fractions the orientation of substrate to the phospholipase A2 is an that were pooled as indicated by the widths of the bars. important determinant of the activity of the enzyme (16) and the orientation of F2-isoprostane-containing phosphatidyl- teristics of phosphatidyicholine species containing F2-iso- choline in the conditions used in vitro, because of its en- prostanes. In addition, fractions also were eluted between 5 hanced polarity, may be quite different from the orientation and 10 min in which free isoprostanes were detected after ofarachidonoyl phosphatidylcholine. However, these results saponification. Although these latter fractions were not fur- do indicate that isoprostane-containing phospholipids can ther analyzed, they were thought likely to represent phos- indeed serve as substrates for phospholipase A2. pholipid species other than phosphatidylcholine and/or neu- Evidence That F2-Isoprostanes Are Not Formed from Free tral lipids that contain esterified F2-isoprostanes. Arachidonic Acid and Incorporated into Lysophospholipids. Analysis of the Polar F2-Isoprostane-Containing Lipids by Although the above findings suggested strongly that F2- Fast Atom Bombardment Mass Spectrometry. The polar lipids isoprostanes exist in ester linkages to lipids in vivo, it could described above that eluted from the HPLC column between not be concluded from these results that they are actually 35 and 50 ml were then subjected to analysis by negative-ion formed in situ from esterified arachidonic acid. Alternatively, fast atom bombardment mass spectrometry. The character- they might be formed from free arachidonic acid and subse- istics of phosphatidylcholine analyzed by negative-ion fast quently incorporated into lysophospholipids. To investigate atom bombardment mass spectrometry have been detailed this possibility, we examined the ability of a rat liver mi- (20, 21). The negative-ion fast atom bombardment mass crosomal CoA-dependent esterification system to incorpo- spectrum of phosphatidylcholine is characterized by high- rate PGF2,, into lysophosphatidylcholine. Under in vitro mass ions representing losses of 15, 60, and 86 Da from the conditions in which arachidonic acid was quantitatively in- molecular ion. These ions are generated from fragmentations corporated into lysophosphatidylcholine (96% + 3%, n = 4), at different sites in the choline moiety. In addition, assign- essentially no incorporation of PGF2< was detected (1.6% + ment of the substituents at the sn-1 and sn-2 positions can be 0.9%, n = 4). made by the presence of intense ions representing the car- Analysis of Lipid Extracts by Normal-Phase HPLC. Collec- boxylate anions of the fatty acids. The two most abundant tively, the results obtained up to this point provided substan- fatty acids at the sn-1 position in rat liver phosphatidylcholine tial indirect evidence that the formation ofF2-isoprostanes in are palmitate and stearate. The mass spectra obtained from vivo occurs in situ on lipids. We then attempted to obtain analysis ofthe polar F2-isoprostane-containing lipids purified direct physical evidence for the existence of F2-isoprostane- by HPLC (Fig. 3) revealed high-mass negative ions predicted containing phospholipids from analysis of lipids extracted for F2-isoprostane-containing phosphatidylcholine at M - 15 from livers of CC14-treated rats by fast atom bombardment (M - CH3)-, M - 60 [M - HN(CH3)31-, and M - 86 [M - mass spectrometry after purification by normal-phase HN(CH3)3C2H2]- for both 1-palmitoyl 2-F2-isoprostane HPLC. Lipid extracts were subjected to normal-phase HPLC phosphatidylcholine and 1-stearoyl 2-F2-isoprostane phos- analysis using a solvent system which separates phosphati- phatidylcholine. As predicted, prominent ions are present at dylcholine from other phospholipids and neutral lipids (17- m/z 255 and 283, representing the carboxylate anions of 19). To detect fractions that contained lipid species contain- palmitate and stearate, respectively, from the sn-i position. ing F2-isoprostanes, aliquots of fractions collected were In addition, and most importantly, there is also a prominent combined and saponified, and free F2-isoprostanes were ion at m/z 353, the predicted ion representing the carboxylate quantified by gas chromatography/mass spectrometry. Frac- anion of F2-isoprostane from the sn-2 position. tions in which the majority of free F2-isoprostanes were Analysis by positive-ion fast atom bombardment mass detected after hydrolysis (68% of the total) eluted at a much spectrometry further confirmed the structure and molecular more polar retention volume (35-50 ml) than nonoxidized weight of these F2-isoprostane-containing species of phos- phosphatidylcholine, which eluted between 18 and 25 ml (Fig. phatidylcholine. In addition to a prominent ion at m/z 184 2). This is consistent with the predicted more polar charac- from the phosphocholine moiety, prominent ions were also Downloaded by guest on September 29, 2021 10724 Pharmacology: Morrow et al. Proc. Natl. Acad Sci. USA 89 (1992)

283 O Rlz (CH2),6CH, (stearoyl) 100 CH3(CH2)O6 C02 HO .Q H2C-O-C- R2= (CH2)14CH3 (palmltoyl)

79 I 90- 'I to HO O \H "HC-O -P-0-CH2CH2N (CH3), CH, + H PO,- I 80' - 508 (R') 255 480 (R') 70 CH3(CH),4 COj-

HO M-60 (R') °06-5 ° r~~~o 789 D 50- M-15 (R') 0 HO OH 844 30 -O-P - O-CH2CH2N(CH3)2 40' H 153 I, >0 0 353/ 50,8 M-86 (R') 816 773 M-15 (R) M-86 (R2) 745 \- 30' 20 M-60 (R2) 771 20- 480

10* I im j J i iiil. ILi . X ==ILI III III iLI II 1, ih UIm iWi I IMA IfWiam IIIII-' 100 200 300 400 500 600 700 800 900 1000 m/z FIG. 3. Negative-ion fast atom bombardment mass spectrum obtained from analysis of polar lipids eluted between 33 and 50 ml in Fig. 2B. present at m/z 854 and 882, representing the [M + Na]+ ions phospholipids, and are released preformed. After their re- of 1-palmitoyl 2-F2-isoprostane phosphatidylcholine and lease, isoprostanes are capable ofexerting biological activity. 1-stearoyl 2-F2-isoprostane phosphatidylcholine, respec- We have previously shown that one of the isoprostanes that tively. can be produced in abundance, 8-epi-PGF2a, is an extremely These species of phosphatidylcholine have also been sub- potent renal vasoconstrictor (8). Recently we found that jected to further analysis by tandem mass spectrometry. markedly increased circulating levels of F2-isoprostanes are Analysis of the collision-induced dissociation of the negative present in patients with hepatorenal syndrome, a disorder in ions generated by fast atom bombardment mass spectrometry which the deterioration in renal function has been attributed firmly established the identity of these lipids as phosphati- to renal vasoconstriction (23). We have also shown that dylcholine with palmitate and stearate at the sn-i position and 8-epi-PGF2, is a potent pulmonary artery vasoconstrictor (24) F2-isoprostane at the sn-2 position (J.D.M., L.J.R., and R. C. and that the vasoconstricting actions of 8-epi-PGF2a in both Murphy, unpublished results). the renal and pulmonary vascular beds are mediated through thromboxane/endoperoxide receptors (24, 25). Interestingly, DISCUSSION whereas 8-epi-PGF2, is a potent agonist of vascular throm- boxane/endoperoxide receptors, we have found that it acts These studies provide direct evidence that F2-isoprostanes primarily as an antagonist of platelet thromboxane/endo- exist in ester linkages ofphospholipids in vivo. Evidence was peroxide receptors (26). also obtained indicating that these compounds are not incor- Among the known and sometimes devastating conse- porated preformed into lysophospholipids but are formed in quences ofoxidant injury are alterations in the physicochem- situ by free radical-catalyzed peroxidation of arachidonic ical properties and function of cellular membranes. This has acid in phospholipids. We have previously shown that F2- been attributed to an accumulation of products of lipid isoprostanes can be formed from free arachidonic acid in peroxidation in lipid bilayers (27). Molecular modeling of vitro (9). However, this is unlikely to be a major contributor phosphatidylcholine with the F2-isoprostane 8-epi-PGF2a at to the formation ofthese compounds in vivo because the vast the sn-2 position reveals it to be a remarkably kinked mole- majority of arachidonic acid is present in vivo in ester cule (Fig. 4). Thus, in addition to the fact that these com- linkages ofphospholipids. In addition, it has been shown that pounds can exert bioactivity once they are released from arachidonic acid in phospholipids is much more readily phospholipids, the formation ofisoprostane-containing phos- oxidized than unesterified arachidonic acid (22). For these pholipid species in settings of oxidant stress would be pre- reasons, F2-isoprostanes are likely formed predominantly in dicted to have profound effects on the fluidity and integrity situ on phospholipids, and free isoprostanes detected in of cellular membranes and thus may contribute in an impor- biological fluids in vivo likely derive from the hydrolysis of tant way to these recognized sequelae of oxidant injury that preformed compounds from phospholipids. The results ofthe can lead to severe cellular dysfunction or death. time course experiment presented in Fig. 1 support this Therefore, the biological significance of the findings re- hypothesis in that increases in levels offree compounds in the ported herein potentially encompasses both the formation of circulation lag behind increases in F2-isoprostanes esterified these compounds on phospholipids, which can alter the in lipids in the liver. physicochemical properties of cell membranes, and the fact These findings introduce the following concept: In direct that once released, these compounds can exert biological contrast to cyclooxygenase-derived prostaglandins, which activity in target tissues. How these two processes are are only formed de novo from free arachidonic acid and are balanced will influence the overall biological consequences not stored, isoprostanes are formed from esterified arachi- which ensue as a result of the formation of these compounds donic acid, exist in a storage depot as acyl moieties of in settings of oxidant injury. Presumably central to the Downloaded by guest on September 29, 2021 Pharmacology: Morrow et al. Proc. Natl. Acad. Sci. USA 89 (1992) 10725 tivity of these various endogenous phospholipases for iso- prostane-containing phospholipids will be of relevance. In summary, we report the finding that F2-isoprostanes are formed in situ on phospholipids in vivo and subsequently released preformed, presumably by phospholipase A2. Fur- ther understanding of factors important in regulating the formation and hydrolysis ofisoprostane-containing phospho- lipids should provide valuable insights into key fundamental processes involved in the pathogenesis of oxidant injury. The skillful technical assistance of Tanya Minton, William Zack- ert, and Brian Nobes was greatly appreciated, as was the help of Amanda Simpson in preparing this manuscript. This work was supported by Grants GM 42056, HL 02499, and ES 02497 from the National Institutes of Health. J.D.M. is a Howard Hughes Medical Institute Physician Research Fellow. 1. Halliwell, B. & Gutteridge, J. M. C. (1990) Methods Enzymol. 186, 1-85. 2. Southorn, P. A. & Powis, G. (1988) Mayo Clin. Proc. 63, 390-408. 3. Ames, B. N. (1983) Science 221, 1256-1264. 4. Harman, D. (1981) Proc. Nat!. Acad. Sci. USA 78, 7124-7128. 5. Gutteridge, J. M. C. & Halliwell, B. (1990) Trends Biochem. Sci. 15, 129-135. 6. Orrenius, S., McConkey, D. J., Bellomo, G. & Nicotera, P. (1989) Trends Pharmacol. Sci. 10, 281-286. 7. Esterbauer, H., Striegl, G., Puhl, H. & Rotheneder, G. (1989) Free Rad. Res. Commun. 6, 67-75. 8. Morrow, J. D., Hill, K. E., Burk, R. F., Nammour, T. M., Badr, FIG. 4. Space-filling molecular model of F2-isoprostane- K. F. & Roberts, L. J. (1990) Proc. Natl. Acad. Sci. USA 87, containing phosphatidylcholine with palmitic acid at the sn-i position 9383-9387. and the F2-isoprostane 8-epi-PGF2, at the sn-2 position. 9. Morrow, J. D., Harris, T. M. & Roberts, L. J. (1990) Anal. Bio- chem. 184, 1-10. regulation of the levels of esterified versus free isoprostanes 10. Burk, R. F. & Lane, J. M. (1979) Toxicol. Appl. Pharmacol. 50, are phospholipases. There are conflicting views on the role of 467-478. 11. Radin, N. S. (1969) Methods Enzymol. 14, 245-248. phospholipase A2 in oxidant injury. Whereas it has been 12. Hughes, H., Smith, C. V., Homing, E. C. & Mitchell, J. R. (1983) thought that the removal of oxidized fatty acids by phospho- Anal. Biochem. 130, 431-436. lipase A2 is important in the repair of oxidized membranes, 13. Bligh, E. G. & Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, it has been reported that inhibitors of phospholipase A2 can 911-917. be protective in some settings of oxidant injury (28, 29). With 14. Karara, A., Dishman, E., Falck, J. R. & Capdevila, J. H. (1991) to the formation of J. Biol. Chem. 266, 7561-7569. regards isoprostanes during oxidant 15. Recknagel, R. 0. (1967) Pharmacol. Rev. 19, 145-196. stress, it is possible to envision that either the effects result- 16. Hazlett, T. L., Deems, R. A. & Dennis, E. A. (1990) Adv. Exp. ing from their formation in lipid bilayers or the biological Med. Biol. 279, 49-84. consequences of their activity in the free state may be more 17. Ellingson, J. G. & Zimmerman, R. L. (1987) J. Lipid Res. 28, detrimental, depending on the situation. In addition, quanti- 1016-1018. fication of F2-isoprostanes is emerging as a valuable tool to 18. Hanras, C. & Penn, J. L. (1991) J. Am. Oil Chem. Soc. 68, 804-807. 19. Brash, A. R., Ingram, C. D. & Harris, T. M. (1987) Biochemistry assess endogenous lipid peroxidation and oxidant status in 26, 5465-5471. vivo in humans (30). Depending on the balance of formation 20. Kayganich, K. & Murphy, R. C. (1991)J. Am. Soc. Mass Spectrom. on phospholipids and their release in free form, it may be 2, 45-54. important to quantify levels of both free compounds and 21. Jensen, N. J., Tomer, K. B. & Gross, M. L. (1986) Lipids 21, compounds esterified to lipids in tissues of interest to accu- 580-586. rately assess total production of further 22. Frei, B., Stocker, R. & Ames, B. N. (1988) Proc. Natl. Acad. Sci. isoprostanes. Thus, USA 85, 9748-9752. understanding of the role of phospholipases in oxidant injury 23. Moore, K. P., Morrow, J. D., Awad, J. A., Ravenscraft, M. D., and specifically in relation to their activity towards isopros- Marini, G., Williams, R. & Roberts, L. J. (1992) Clin. Res. 40, 313A. tane-containing phospholipids will be important. 24. Banerjee, M., Kang, K. H., Morrow, J. D., Roberts, L. J. & In this context, the type(s) of phospholipase A2 important Newman, J. H. (1992) Am. J. Physiol., in press. in hydrolyzing F2-isoprostanes in vivo is at present not 25. Takahashi, K., Nammour, T. M., Fukunaga, M., Ebert, J., Mor- known. Previous studies have shown that oxidized row, J. D., Roberts, L. J., Hoover, R. L. & Badr, K. F. (1992) fatty acids J. Clin. Invest. 90, 136-141. esterified in phospholipids are preferential substrates for low 26. Morrow, J. D., Minton, T. A. & Roberts, L. J. (1992) Prostoglan- molecular weight phospholipase A2 (27). In this regard, we dins 44, 155-163. demonstrated that F2-isoprostane-containing phospholipids 27. Sevanian, A. & Kim, E. (1985) J. Free Rad. Biol. Med. 1, 263-271. are substrates for the low molecular weight phospholipase A2 28. Otamiri, T. & Tagesson, C. (1989) Am. J. Surg. 157, 562-566. in bee venom, which is related to mammalian low molecular 29. Chiarielio, M., Ambrosio, G., Cappelli-Bigazzi, M., Nevola, E., Perrone-Filardi, P., Marone, G. & Condorelli, M. (1987) J. Phar- phospholipases (31). In contrast, the high molecular weight macol. Exp. Ther. 241, 560-568. phospholipase A2 recently characterized by Clark et al. (32) 30. Morrow, J. D. & Roberts, L. J. (1991) J. Free Rad. Biol. Med. 10, exhibits a marked specificity for hydrolyzing arachidonic 195-200. acid linkages compared with those of other unsaturated fatty 31. Cordella-Miele, E., Miele, L., Beninati, S. & Mukherjee, A. B. acids at the sn-2 position. On the other hand, platelet- (1990) Adv. Exp. Med. Biol. 279, 105-123. activating factor acetylhydrolase exhibits low 32. Clark, J. D., Lin, L. L., Kriz, R. W., Ramesha, C. S., Sultzman, only activity L. A., Lin, A. Y., Milona, N. & Knopf, J. L. (1991) Cell 65, for hydrolysis of esters of nonoxidized long chain fatty acids 1043-1051. but will hydrolyze esters of longer chain fatty acids that have 33. Stremler, K. E., Stafforini, D. M., Prescott, S. M. & McIntyre, been oxidized (33). Thus, future studies examining the ac- T. M. (1991) J. Biol. Chem. 266, 11095-11103. Downloaded by guest on September 29, 2021